Phage display of antibodies was initially based on systems developed for the display of peptides (Smith, Science 228, 1315-7, 1985). Antibody single chain variable domains were fused to the coat protein gene (gpIII), (McCafferty, et al., Nature 348, 552-554, 1990) resulting in all the gpIII molecules displaying fusion antibodies. However, the fusion of a polypeptide to the gpIII reduced the ability of the phage to infect bacteria and secondly the multivalent display at the tip of the phage resulted in avidity selection rather than affinity discrimination. Utilizing a phagemid vector (to present the gpIII-fusion) and helper phage rescue (to introduce the wild type gpIII), the valency of fusion display was reduced and infectivity restored (Bass, et al, Proteins: Structure, Function, and Genetics 8, 309-314, 1990). Likewise, the display of heterodimeric polypeptides such as antibody F(ab) fragments as either major (gpVIII) (Kang, et al., Proc. Natl Acad. Sci. USA 88, 4363-66, 1991) , or minor (gpIII) (Barbas, et al., Proc. Natl. Acad. Sci. USA 88, 7978-82, 1991; Garrard, et al., Bio/Technol. 9, 1373-77, 1991) coat protein fusions has successfully utilized phagemid with helper phage rescue.
Phage display of antibody fragments and other polypeptides has gained acceptance as a useful tool in contemporary molecular immunology. The density of polypeptide display per filamentous phage particle is influenced by the choice of which phage coat protein is used as fusion partner and the type of vector system used. Molecules expressed from nucleotide sequences fused with the sole copy of gpIII on the phage genome such as fd or M13 resulted in a multivalent cluster display (tri-penta valent) and reduced infectivity of bacteria (McCafferty et al., Nature 348, 552-554, 1990; Smith, Science 228, 1315-7, 1985). Multivalent binding of phage with ligand would favor avidity selection and limit the ability to discriminate between modest gains in affinity (Cwirla, et al., Proc. Natl Acad. Sci. USA 87, 6378-82., 1990). This may be desirable when attempting to isolate ligand binding molecules of lower affinity. Phagemid vectors encoding phage coat protein fusion polypeptides used in conjunction with helper phage rescue, generated phage with restored infectivity and reduced valency permitting enrichment for high affinity interactions (Bass, S., et al. Proteins: Structure, Function, and Genetics 8, 309-314, 1990).
Both the high and low density display systems have uses in accessing ligands against target receptors or tissues. It would be desirable to create a phage display system in which the density of the displayed fusion moieties on the phage particle could be modulated from a few displayed copies to less than 1 per phage. To achieve this with existing vectors requires shuttling of inserts between gpIII phage and gpVIII/gpIII phagemid vectors. However this may also be attained by utilizing a single M13 phage based vector with a synthetic second copy of the gene encoding gpIII or gpVIII (i.e. pseudo wild type) as a fusion partner (Huse, et al., J. Immunol. 149, 3914-20, 1992), and manipulating the phage growth conditions to favor low or moderate rates of fusion incorporation into the phage filament. Incorporating the display expression cassette onto the phage genome may also have the added benefits of fusion expression being synchronous with phage morphogenesis. The present invention describe a phage vector in which a polypeptide is displayed on the phage surface. This display system was used to investigate the modulation of display fusion on phage resulting in optimal phage display as determined by relative panning enrichment efficacy.